Mella Jessica, Volk Regan F, Zaro Balyn W, Buchwalter Abigail
Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States.
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, United States.
Elife. 2025 Aug 28;14:RP105937. doi: 10.7554/eLife.105937.
The inner nuclear membrane (INM), a subdomain of the endoplasmic reticulum (ER), sequesters hundreds of transmembrane proteins within the nucleus. We previously found that one INM protein, emerin, can evade the INM by secretory transport to the lysosome, where it is degraded (Buchwalter et al., 2019). In this work, we used targeted mutagenesis to identify intrinsic sequences that promote or inhibit emerin's secretory trafficking. By manipulating these sequences across several tag and expression level combinations, we now find that emerin's localization is sensitive to C-terminal GFP tagging. While emerin's long, hydrophobic C-terminal transmembrane domain facilitates trafficking to the lysosome, extending its lumenal terminus with a GFP tag biases the protein toward this pathway. In contrast, we identify a conserved ER retention sequence that stabilizes N- and C-terminally tagged emerin by limiting its lysosomal flux. These findings underscore long-standing concerns about tagging artifacts and reveal novel determinants of tail-anchored INM protein targeting.
内核膜（INM）是内质网（ER）的一个亚结构域，它在细胞核内隔离了数百种跨膜蛋白。我们之前发现，一种内核膜蛋白emerin可以通过分泌运输到溶酶体而避开内核膜，并在那里被降解（Buchwalter等人，2019年）。在这项工作中，我们使用靶向诱变来鉴定促进或抑制emerin分泌运输的内在序列。通过在几种标签和表达水平组合中操纵这些序列，我们现在发现emerin的定位对C端GFP标签敏感。虽然emerin长的疏水C端跨膜结构域有助于其运输到溶酶体，但用GFP标签延伸其腔端会使该蛋白偏向于这条途径。相比之下，我们鉴定出一个保守的内质网保留序列，该序列通过限制其溶酶体通量来稳定N端和C端标记的emerin。这些发现强调了长期以来对标签假象的担忧，并揭示了尾锚定内核膜蛋白靶向的新决定因素。
